6. PROJECT SUMMARY/ABSTRACT Most vital organs in the body (Kidney, Intestine, Liver, Lung, Glands) are composed of epithelial cells which carry out the main functions of the organ, necessary for survival of the organism. Normal epithelial cell function depends on a fundamental property of these cells, polarity, i.e., the ability to segregate their surface proteins (e.g. metabolite transporters, proteolytic enzymes, hormone receptors) to either the apical or the basolateral domains of the plasma membrane, which are separated by an elaborate junctional complex. The long term objective of this proposal is to elucidate the molecular mechanisms responsible for the establishment and maintenance of polarized trafficking routes to the cell surface. The experiments proposed will utilize state of the art biochemical polarity assays and quantitative live imaging techniques developed by our laboratory to elucidate these trafficking mechanisms in the model epithelial cell lines MDCK (derived from distal kidney tubule), LLC-PK1 and IRPT (derived from proximal kidney tubule). Specific aims 1 and 2 will investigate in detail the roles of clathrin and clathrin adaptors in apical-basolateral protein sorting, as our preliminary work suggests the hypothesis that these molecules play a key role in the sorting of basolateral proteins. We will inhibit the expression or function of clathrin and clathrin adaptors using techniques already available in our laboratory (siRNA, X-linking, function-blocking antibodies) to study their sorting role at the level of Trans Golgi Network (TGN) and Recycling Endosomes (RE), the main sorting compartments in epithelial cells. We will extend these studies to the kidney nephron, as our preliminary data indicate that the best characterized epithelial sorting adaptor, AP1B, is absent in the proximal convoluted tubule (PCT);we will test the hypothesis that lack of AP1B facilitates apical endocytosis and recycling of receptors (e.g. Megalin) necessary for retrieval of proteins from the glomerular ultrafiltrate. Specific aims 3 and 4 will study in detail the fundamental role of the actin cytoskeleton in the generation of basolateral and apical transport routes. Specific aim 3 will focus on the actin organizers cdc42 and IQGAP1, as our preliminary work indicates these molecules play a key role in basolateral delivery, similar to their crucial role in the yeast secretory route, which appears to be conserved in the mammalian basolateral routes. Specific aim 4 will focus on the roles of Syndapins, dynamin 2 and LIMK1, as our preliminary data suggests their crucial participation in apical protein exit routes from the TGN. Because epithelial polarity is involved in key development processes and because its derangement is a cause of cancer and many diseases of kidney and other epithelial organs, the experiments proposed are bound to generate important information relevant to Human Health and Disease. 7. PROJECT NARRATIVE Most vital organs in the body (Kidney, Intestine, Liver, Lung, Glands) are composed of epithelial cells which carry out the main functions of the organ, necessary for survival of the organism. Normal epithelial cell function depends on a fundamental property of these cells, polarity, i.e., the ability to segregate their surface proteins (e.g. metabolite transporters, proteolytic enzymes, hormone receptors) to either the apical or the basolateral domains of the plasma membrane, which are separated by an elaborate junctional complex. The long term objective of this proposal is to elucidate the molecular mechanisms responsible for the establishment and maintenance of polarized trafficking routes to the cell surface, using state of the art biochemical polarity assays and quantitative live imaging techniques developed by our laboratory, in model kidney epithelial cell lines. Some of the experiments will study how these mechanisms may explain the function of the proximal convoluted tubule (PCT) of the kidney. Because epithelial polarity is a key for many developmental processes and because derangement of polarity is a cause of cancer and many diseases of kidney and other epithelial organs, the experiments proposed are bound to generate important information relevant to Human Health and Disease.